Project Summary Diabetes and non-alcoholic fatty liver disease (NAFLD) are closely associated with hepatic fat and cholesterol accumulation, hepatocyte organelle dysfunction, low grade inflammation, and dyslipidemia. Patients with diabetes and NAFLD have significantly higher risk of cardiovascular disease, which remains the leading cause of death worldwide. Hepatic bile acid synthesis is the only major cholesterol catabolism mechanism in the body. Furthermore, bile acids act as signaling molecules to critically control metabolic homeostasis and inflammatory response. Different therapeutic approaches targeting the bile acid dynamics and signaling pathways have shown great promise for treating metabolic and chronic liver diseases. However, how modulating the enterohepatic bile acid signaling impacts the complex metabolic network via distinct mechanism of actions is still incompletely understood. New mechanistic insights are clearly needed to establish the molecular basis for developing effective bile acid-based therapies. The goal of this study is to define a new role of Transcriptional Factor EB (TFEB) in the regulation of hepatic bile acid metabolism. TFEB belongs to the basic helix-loop-helix leucine zipper family of transcriptional factors and was recently identified as a nutrient and stress-sensing master regulator of lysosomal biogenesis in various cell types, which has led to a paradigm shift in our understanding of how lysosomal pathways can be dynamically regulated in response to various nutrient and stress signals to maintain cellular homeostasis. Current studies suggest that TFEB may be an attractive target for treating neurodegenerative diseases, lysosomal storage disease, and metabolic diseases. However, the role of TFEB in regulating the complex hepatic metabolism is incompletely understood, and the TFEB regulation of hepatic bile acid metabolism has not been explored. Here we found that TFEB is a strong inducer of bile acid synthesis, and hepatic TFEB itself is inhibited by the intestine bile acid sensing hormone FGF15/19. Identification of this gut-liver signaling crosstalk has led to an interesting new concept of pharmacologically targeting this regulatory loop to enhance hepatic TFEB function and improve metabolic homeostasis. Three specific aims are designed to first establish that liver TFEB is a novel bile acid sensing transcriptional factor and a key effector in the gut-liver bile acid signaling crosstalk regulation of liver metabolism, and further determine the pathophysiological significance and therapeutic implications of this signaling regulatory loop. This study will employ several experimental mouse models through AAV8-mediated hepatocyte-specific gene delivery, tissue-specific genetic knockout, and pharmacological treatment approaches. These models will be investigated with a combination of physiological, molecular biology, and cell biology techniques and unbiased proteomics approaches. By employing these state-of-the-art models and techniques, our working hypothesis can be rigorously tested and new mechanistic insights will be obtained.